We Claim:
1. A method for purifying a multicomponent sample by reverse phase chromatography
comprising:

(a) configuring a chromatographic system having a hydrophobic stationary phase;

(b) saturating the chromatographic stationary phase with quaternary ammonium salt or quaternary phosphonium salt;

(c) optionally washing the column after the step (b) with a buffer;

(d) applying a multicomponent sample to one end of the chromatographic bed
comprising stationary phase with hydrophobic quaternary ammonium salt or quaternary
phosphonium salt;

(e) eluting the muticomponent sample in a buffer;

(f) recovering the desired component of the sample.

2. The method of claim-1, wherein said quaternary ammonium salt has the structure as
mentioned below- R3 wherein R, R , R , R ís selected independently from the group comprising straight or branched alkyl, cyclic hydrocarbons, aromatic group, alkyl substituted aromatic group, aryl substituted alkyl groups; and B denotes an anión.

3. The method of claim-2, wherein said quaternary ammonium salt is selected from the group comprising of bis(trifluoromethylsulfonyl)imide, bis(fluorosulfonyl)imide, dicyanamide,
halogens, tetrafluoroborate, hexafluorophosphate, trifluoromethanesulfonate, methanesulfonate,
trifluoroacetate, thiocyanate, dimethylphosphate, diethylphosphorodithioate, amino acids, terta
butyl ammonium bromide, tetra-butyl-ammonium hydrogen sulfate, tetra-butyl-ammonium
hydroxide, tetra-octyl-ammonium bromide, methyl-trioctyl-ammonium chloride, myristyl
trimethyl ammonium bromide and cetyl trimethyl ammonium chloride.

4. The method of claim-1, wherein said quaternary phosphonium salt has the structure as
mentioned below- R3 wherein R, R1, R2, R3 is selected independently from the group comprising straight or branched alkyl, cyclic hydrocarbons, aromatic group, alkyl substituted aromatic group, aryl substituted alkyl groups; and B denotes an anión.

5. The method of claim-4, wherein said quaternary ammonium salt is selected from the group
comprising bis(trifluoromethylsulfonyl)imide, bis(fluorosulfonyl)imide, dicyanamide, halogens,
tetrafluoroborate, hexafluorophosphate, trifluoromethanesulfonate, methanesulfonate,
trifluoroacetate, thiocyanate, dimethylphosphate, diethylphosphorodithioate, ethyl-triphenyl-
phosphonium bromide, ethyl-triphenyl-phosphonium-iodide, butyl-triphenyl-phosphonium
bromide, methyl-triphenyl-phosphonium bromide, triphenyl phosphoium bromide and butyl-
triphenyl-phosphonium-chloride.

6. A method for purifying a multicomponent sample by reverse phase chromatography
comprising:

(a) configuring a chromatographic system having a hydrophobic stationary phase;

(b) saturating the chromatographic stationary phase with quaternary ammonium salt or quaternary phosphonium salt;

(c) optionally washing the column after the step (b) with a buffer;

(d) applying a multicomponent sample to one end of the chromatographic bed comprising stationary phase with hydrophobic quaternary ammonium salt or quaternary phosphonium salt;

(e) eluting the multicomponent sample in a buffer containing quaternary ammonium salt or quaternary phosphonium salt; and

(í) recovering the desired component of the sample.

7. A method for purifying a multicomponent sample by reverse phase chromatography comprising:

(a) confíguring a chromatographic system having a hydrophobic stationary phase;

(b) saturating the chromatographic stationary phase with quatemary ammonium salt or quatemary phosphonium salt;

(c) optionally washing the column after the step (b) with a buffer;

(d) applying a multicomponent sample to one end of the chromatographic bed comprising stationary phase with hydrophobic quatemary ammonium salt or quatemary phosphonium salt;

(e) eluting the multicomponent sample in a buffer;

(f) recovering the desired component of the sample;

(g) treating the equilibrated the chromatographic stationary phase with quatemary ammonium salt or quatemary phosphonium salt with sodium tetrafluoroborate; and

(h) washing the treated chromatographic stationary phase after step (g) with a solvent to recover the chromatographic stationary phase from the quatemary ammonium salt or quatemary phosphonium salt.

FIELD OF THE INVENTION
The invention relates to purification of organic compounds using surrogate stationary phases on reversed phase columns. More particularly, the invention provides a preparative HPLC method for purification of organic compounds employing reagents selected from hydrophobic quaternary ammonium salt or quaternary phosphonium salt as a surrogate stationary phase.
BACKGROUND OFTHE INVENTION
Reversed phase high performance liquid chromatography (RP-HPLC) is used ubiquitously in academic institutions, forensic laboratories, fine chemicals, and pharmaceutical industries etc. for the analysis, characterization, separation, purification and/or isolation of small organic molecules, natural products, and biologically active molecules such as polypeptides, proteins, and nucleotides. In the pharmaceutical industry, analytical RP-HPLC is used for the reléase and characterization of raw materials, intermediates, and active pharmaceutical ingredients (APIs). Preparative reversed phase high performance liquid chromatography (Prep-RP-HPLC) is used for the commercial production of Peptide APIs, and most other complex APIs that are not amenable to crystallization.
Preparative RP-HPLC in the elution mode is limited by the loading capacity of the analyte. In the elution preparative RP-HPLC mode, the typical loading capacity of synthetic peptides is in the range of 1 to 2 mgs per mi of packed column volume (viz., 0.1% to 0.2% with respect to total column volume).
The patent application US20120322976 discloses a preparative HPLC of a GLP-1 analog. The loading was 0.225% with respect to total column volume {(about 45 mgs on to a 20 mi C-18 substituted (Octadecyl-dimethylsilyl) silica resin (particle size: 15 microns)}.
The patent application US20110313131 discloses a preparative HPLC of (Aib 8, 35) GLP-1 (7-36)-NH2 at loadings up to 20 g/ L (2% with respect to total column volume).
2

Recent advances in RP-HPLC have focussed on producing spherical silica and development of new bonding chemistries to furnish stationary supports that have improved stability and selectivity. The earlier supports were irregular silica particles that were derivatized with C-18 or C-8 chains, and they suffered from high back pressure. The high back pressure limited their use with respect to quantity that could be purifíed in a single run, and to relatively smaller diameter columns.
The commercial manufacture of spherical silica that has been derivatized by C-18, C-8, and other ligands have overeóme these challenges and have extended the utility of preparative HPLC vastly. These technological advances in process HPLC instrumentation, and the bonded silica supports have made possible commercial production of complex peptides such as Fuzeon®, a 36-amino acid peptide, in ton quantities. Unfortunately, these large scale HPLC instruments and the associated column hardware are very costly and restrict the affordability of the methods.
Further, RP-HPLC in the displacement mode has better loading capacity than RP-HPLC in the elution mode but it is arduous to develop. The displacement is best suited for ion exchange mode, and has found numerous recent applications.
Displacement chromatography, on the other hands, utilizes as mobile phase a displacer solution which has higher affinity for the stationary phase material than do the sample components. The key operational feature which distinguishes displacement chromatography from elution chromatography is the use of a displacer molecule.
The Patent US6239262 discloses low molecular weight displacers for protein purification in hydrophobic interaction and reverse phase chromatographic systems.
The PCT publication WO2013052539 discloses the process for separating organic compounds from a mixture by reverse-phase displacement chromatography, including providing a hydrophobic stationary phase; applying to the hydrophobic stationary phase a mixture comprising organic compounds to be separated; displacing the organic compounds from the hydrophobic stationary phase by applying thereto an aqueous composition comprising a non-
surface active hydrophobic cationic displacer molecule and about 10% wt or less of an organic
3

solvent; and collecting a plurality of fractions eluted from the hydrophobic stationary phase containing the organic compounds; in which the non-surface active hydrophobic cationic displacer molecule comprises a hydrophobic catión and a counterion.
The PCT publication WO2013052087 discloses a process for separating organic compounds from a mixture by reverse phase displacement chromatography, including, providing a hydrophobic stationary phase; applying, to the hydrophobic stationary phase a mixture comprising organic compounds to be separated; displacing the organic compounds from the hydrophobic stationary phase by applying thereto an aqueous composition comprising a non-surface active hydrophobic neutral zwitterionic displacer molecule and optionally an organic solvent; collecting a plurality of fractions eluted from the hydrophobic stationary phase containing the separated organic compounds.
In displacement chromatography separations, the sample components are introduced in the form of homogeneous sample solution, so that individual components are each delivered at a constant concentration throughout the sample application step. The driving forcé for separation is that weak binders are displaced from the limited number of binding sites on the stationary phase material by more strongly binding bulk of the product. This proceeds in a continuous manner until the product and other stronger binders are fully retarded in the earlier part of the chromatography bed, thus permitting the more weakly binding impurities to stay bound to the stationary phase material further along the chromatography bed. Once all sample molecules are bound to the stationary phase, no further movement of these molecules will be observed. A problem which may occur because of such use of homogeneous sample solutions, however, is that molecules of strongly binding components introduced during an early part of sample application may inadvertently be displaced by weaker binders introduced during a later stage of sample application.
The patent US6576134 overcomes the above problem in displacement chromatography by applying the sample components to the chromatography bed in a non-homogeneous manner such that the concentration of at least one component with relatively low affinity for the stationary phase material is enhanced during an earlier part of sample application and/or the concentration
4

of at least one component with relatively high affinity for the stationary phase material is enhanced during a later part of sample application.
Therefore there is a need for a simple, cost effective and scalable separation process for peptides by employing preparative reverse HPLC method.
OBJECTS OF THE INVENTION
The primary object of the invention is to provide a preparative HPLC method for purification of organic compounds.
Another object of the invention is to provide a preparative HPLC method for purification of organic compounds employing quatemary ammonium salt or quatemary phosphonium salt as a surrogate stationary phase.
A further object of the invention is to provide a simple, cost effective and scalable separation method for peptides by employing preparative HPLC method.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a method for purifying a multicomponent sample by reverse phase chromatography comprising:
(a) configuring a chromatographic system having a hydrophobic stationary phase;
(b) saturating the chromatographic stationary phase with quatemary ammonium salt or quatemary phosphonium salt;
(c) optionally washing the column after the step (b) with a buffer; and
(d) applying a multicomponent sample to one end of the chromatographic bed comprising stationary phase with hydrophobic quatemary ammonium salt or quatemary phosphonium salt; and
(e) eluting the muticomponent sample in a buffer;
(f) recovering the desired component of the sample.
5

In another aspect, the t invention provides a method for purifying a multicomponent sample by reverse phase chromatography comprising:
(a) configuring a chromatographic system having a hydrophobic stationary phase;
(b) saturating the chromatographic stationary phase with quaternary ammonium salt or quaternary phosphonium salt;
(c) optionally washing the column after the step (b) with a buffer;
(d) applying a multicomponent sample to one end of the chromatographic bed
comprising stationary phase with hydrophobic quaternary ammonium salt or
quaternary phosphonium salt;
(e) eluting the multicomponent sample in a buffer containing quaternary ammonium salt or quaternary phosphonium salt; and
(f) recovering the desired component of the sample.
In yet another aspect, the invention provides a method for purifying a multicomponent sample by reverse phase chromatography comprising:
(a) configuring a chromatographic system having a hydrophobic stationary phase;
(b) saturating the chromatographic stationary phase with quaternary ammonium salt or quaternary phosphonium salt;
(c) optionally washing the column after the step (b) with a buffer;
(d) applying a multicomponent sample to one end of the chromatographic bed comprising
stationary phase with hydrophobic quaternary ammonium salt or quaternary
phosphonium salt;
(e) eluting the muticomponent sample in a buffer;
(í) recovering the desired component of the sample;
(g) treating the equilibrated chromatographic stationary phase with quaternary
ammonium salt or quaternary phosphonium salt with sodium tetrafluoroborate; and
(h) washing the treated chromatographic stationary phase after step (g) with a solvent to recover the chromatographic stationary phase from the quaternary ammonium salt or quaternary phosphonium salt. Still another aspect of the invention is to provide a preparative HPLC method for purification of organic compounds wherein the method has following advantages (1) increased loading (2)
6

limited use of solvents (3) reduced waste disposal (4) ease of operation, and (5) lower scale of the equipments.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig-1: Preparative HPLC data for the purification of Leuprolide acétate of the present invention
by Discovery Bio Wide Pore (10 mm X 250 mm, C18, 5u,300Á pore diameter Fig-2: Preparative HPLC data for the purification of Leuprolide acétate of the present invention
by Waters Symmetry (19 mm X 50 mm, C8, 5u, 120 Á pore diameter Fig-3: Preparative HPLC data for the purification of Leuprolide acétate by Standard preparative
HPLC by the column YMC, ODS-AQ (50 mm X 250 mm, C18, 10 u, 120 Á pore
diameter [Comparative example] Fig-4: Preparative HPLC data for the purification of Leuprolide acétate of the present invention
by employing n-Tetrabutylammonium bromide (TBA-Br) Fig-5: Preparative HPLC data for the purification of Leuprolide acétate of the present invention
by employing n-tetrabutylammonium hydrogen sulfate (TBA-HS) Fig-6: Preparative HPLC data for the purification of Leuprolide acétate of the present invention
by employing Cetyltrimethylammonium bromide (CTA-Br) Fig-7: Preparative HPLC data for the purification of Leuprolide acétate of the present invention
by employing n-Tetrabutylphosphonium chloride (TBP-C1) Fig-8: Preparative HPLC data for the purification of Leuprolide acétate of the present invention
by employing n-Tetrabutylammonium chloride (TBA-C1)
DETAILED DESCRIPTION OF THE INVENTION
First embodiment of the present invention provides a preparative HPLC method for purification of organic compounds employing quaternary ammonium salt as a surrogate stationary phase, wherein the chromatographic stationary phase is hydrophobic.
7

The quaternary ammonium salt of the present invention has the structure as mentioned below:
R B"
R=—N-—R1
R3
wherein R, R , R , R is selected independently from the group comprising straight or branched alkyl, cyclic hydrocarbons, aromatic group, alkyl substituted aromatic group, aryl substituted alkyl groups; the anión denoted as B herein in the compound represented by the formula (1) includes bis(trifiuoromethylsulfonyl)imide, bis(fluorosulfonyl)imide, dicyanamide, halogens, tetrafiuoroborate, hexafluorophosphate, trifluoromethanesulfonate, methanesulfonate, trifluoroacetate, thiocyanate, dimethylphosphate, diethylphosphorodithioate, amino acids, tetra butyl ammonium bromide, tetra-butyl-ammonium hydrogen sulfate, tetra-butyl-ammonium hydroxide, tetra-octyl-ammonium bromide, methyl-trioctyl-ammonium chloride, myristyl trimethyl ammonium bromide and cetyl trimethyl ammonium chloride. Preferably tetra-butyl-ammonium hydrogen sulphate.
Second embodiment of the present invention provides a preparative HPLC method for purification process of organic compounds employing quaternary phosphonium salt as a surrogate stationary phase in hydrophobic stationary phases, preferably C-18, C-4 and C-8 hydrophobic stationary phase.
The quaternary phosphonium salt of the present invention has the structure as mentioned below,
R B"
R2— P+— R1
R3 wherein R, R1, R2, R3 is selected independently from the group comprising straight or branched alkyl, cyclic hydrocarbons, aromatic group, alkyl substituted aromatic group, aryl substituted alkyl groups; the anión denoted as B herein in the compound represented by the formula (1) includes bis(trifluoromethylsulfonyl)imide, bis(fluorosulfonyl)imide, dicyanamide, halogens, tetrafiuoroborate, hexafluorophosphate, trifluoromethanesulfonate, methanesulfonate,

trifluoroacetate, thiocyanate, dimethylphosphate, diethylphosphorodithioate, ethyl-triphenyl-phosphonium bromide, ethyl-triphenyl-phosphonium-iodide, butyl-triphenyl-phosphonium bromide, methyl-triphenyl-phosphonium bromide, triphenyl phosphonium bromide, butyl-triphenyl-phosphonium-chloride.
According to the process of the invention, the concentration of the organic modifíer is held at a sufficiently low concentration to ensure/ enforce strong binding of the analytes to the stationary phase (s).
The surrogate stationary phase in the present invention refers to a modified hydrophobic stationary phase resulted after equilibrating the chromatographic hydrophobic stationary phase with quaternary ammonium salt or quaternary phosphonium salt.
The method of the present invention for purifying a peptide by reverse phase chromatography involves the step of applying to the hydrophobic stationary phase a mixture comprising organic compounds to be separated after the addition of the displacer with or without the organic modifíer, whereas the reverse phase displacement chromatography as disclosed in the Patent US6239262, PCT publications WO2013052539 and WO2013052087, for separating organic compounds from a mixture involves the step of applying to the hydrophobic stationary phase a mixture comprising organic compounds to be separated before the addition of the displacer with or without the organic modifíer.
In various embodiments, the gradient elution can be accomplished, for example, stepwise, linearly, with multi segmented linear or stepwise changes in composition, or with a combination thereof. In one aspect, gradient elution is performed in increasing amounts of an organic modifíer and elution is completed in greater than about 10%, greater than about 20%, greater than about 30%, greater than about 90%>, or up to and including about 100% of the organic modifíer. In certain aspects, elution is completed in decreasing amount of organic modifíer, e.g., less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2%, less than about 1% or about 0% of organic modifíer.
9

The organic modifier in the present invention refers to a solvent or a compound which can be used in chromatographic procedures and like separation methods, to alter the properties of the mobile phase to controllably effect serial elution of desired materials. In one aspect, an organic modifier decreases ionic interactions between molecules in the mobile phase and the stationary phase. For example, in one aspect, an organic modifier comprises a solvent added to a mobile phase to decrease its polarity. Suitable organic modifiers include, but are not limited to, acetonitrile, ethanol, methanol, ethanol, n-propanol or iso-propanol. The separating can be accomplished with any suitable solvent or solvent combination.
The nature of the library useful in the system essentially is unlimited. Thus, mixtures of organic compounds may be used. Digests of biopolymers, either natural or synthetic, are particularly attractive. Such digests may comprise mixtures of peptides, polysaccharides, polynucleotides, various derivatized forms thereof, and variously sized fragments thereof. The biopolymers may be extracted from plant or animal tissues, diseased or healthy, digested if necessary, or used as is. Such librarles are available in abundance, easy to prepare, may be of lower toxicity and more stable than synthetic peptides, and may be varied and screened systematically.
In an embodiment, the concentration of the quaternary ammonium salt or hydrophobic quaternary phosphonium salt in the organic modifier is increased to effect elution of the analytes. The organic modifier may be used with or without the quaternary ammonium salt or the hydrophobic quaternary phosphonium salt.
Third embodiment of the present invention is to provide a process for the removal of the reagents such as hydrophobic quaternary ammonium salt or quaternary phosphonium from the C-18 or C-8 column by employing sodium tetrafiuoroborate or potassium hexafiuorophosphate with organic modifier.
The fourth embodiment of the present invention is to provide a method for purifying a multicomponent sample by reverse phase chromatography comprising:
(a) configuring a chromatographic system having a hydrophobic stationary phase;
(b) saturating the chromatographic stationary phase with quaternary ammonium salt or
quaternary phosphonium salt;
10

(c) optionally washing the column after the step (b) with a buffer;
(d) applying a multicomponent sample to one end of the chromatographic bed comprising stationary phase with hydrophobic quatemary ammonium salt or quatemary phosphonium salt; and

(e) eluting the muticomponent sample in a buffer; and
(f) recovering the desired component of the sample.
In another aspect, the invention provides a method for purifying a multicomponent sample by reverse phase chromatography comprising:
(a) configuring a chromatographic system having a hydrophobic stationary phase;
(b) saturating the chromatographic stationary phase with quatemary ammonium salt or quatemary phosphonium salt;
(c) optionally washing the column after the step (b) with a buffer;
(d) applying a multicomponent sample to one end of the chromatographic bed comprising stationary phase with hydrophobic quatemary ammonium salt or quatemary phosphonium salt;
(e) eluting the multicomponent sample in a buffer containing quatemary ammonium salt or quatemary phosphonium salt; and
(f) recovering the desired component of the sample.
In yet another aspect, the invention provides a method for purifying a multicomponent sample by reverse phase chromatography comprising:
(a) configuring a chromatographic system having a hydrophobic stationary phase;
(b) saturating the chromatographic stationary phase with quatemary ammonium salt or quatemary phosphonium salt;
(c) optionally washing the column after the step (b) with a buffer;
(d) applying a multicomponent sample to one end of the chromatographic bed
comprising stationary phase with hydrophobic quatemary ammonium salt or quatemary
phosphonium salt;
(e) eluting the muticomponent sample in a buffer;
(f) recovering the desired component of the sample;
(g) treating the equilibrated the chromatographic stationary phase with quatemary
11

ammonium salt or quaternary phosphonium salt with sodium tetrafluoroborate; and (h) washing the treated chromatographic stationary phase after step (g) with a solvent to recover the chromatographic stationary phase from the quaternary ammonium salt or quaternary phosphonium salt.
Conventional RPLC hardware systems may be used for the separation, and the term "configuring a chromatographic system" refers to setting up a column or system of column, pump and detector as well known in the art.
The term "saturating the chromatographic stationary phase" refers to passing the quaternary ammonium salt or quaternary phosphonium salt in a solution over the stationary phase in a particular concentration, thereby preparing the surrogate stationary phase.
In a preferred embodiment of the invention, wherein preparative HPLC method for purifícation of organic compounds maintains the concentration of the organic modifier at low, to retain the surrogate stationary phase on the column. The said conditions are required for the interaction of surrogate stationary phase with solute along with interaction with C-18, C-4 and C-8 ligands.
Some aspects and embodiments of this disclosure are described in the examples below, which are provided only for the purpose of illustration and are not intended to limit the scope of the disclosure in any manner.
ILLUSTRATIVE EXAMPLE OF THE PRESENT INVENTION
The C-18/ C-8 reversed phase column is equilibrated with 5 to 10 column volumes (Vcs) of 5 to
10% aqueous acetonitrile containing 10 raM TBAHS. The pH of the starting buffer was not
adjusted, and was about 1.95 (It is important to keep the concentration of acetonitrile lower than
the concentration needed to elute the product on an analytical HPLC column). The crude API
was dissolved starting buffer A or aqueous TFA or aqueous HOAc and loaded on to the column.
After the loading is complete, the column is equilibrated with 2 Column Vcs of Buffer A. Next,
the gradient elution process is started. The buffer B is usually 300 mM to 500 mM TBAHS in 5
to 10% aqueous acetonitrile. A linear gradient of 0%B to 100% Buffer B over 10 Vcs is applied.
12

When the product of interest (API) is about to elute, a gradient hold may be applied until all the API has eluted from the column. Alternately if it is desired to elute the product in a concentrated form the gradient may be allowed to run its course. The fractions containing the puré API product are combined after confirming that the pooled fraction meets the purification criteria. The approximate quantity of the associated TBAHS is calculated. This is then treated with 1.5 to 2 equivalents of sodium tetrafiuoroborate (NaBF4) and extracted with 3 times with chloroform. The aqueous residue is then loaded on to a C-18/ C-8 column from which all the TBAHS has been removed. Removal of TBAHS from the C-18/ C-8 column is accomplished by the following steps: The column is first washed with at least 3 Vcs of 80% Acetonitrile-20% Water. Next, the column is washed with 3Vcs of 100 mM NaBF4 in 80% Acetonitrile-20% water. The column is equilibrated with 1M Acetic Acid in 1% Aqueous Acetonitrile (10 Vcs). The aqueous phase containing "puré API" and excess NaBF4 is diluted with water (5X its volume) and loaded on to the C-18/ C-8 column on to the column. The column is washed with 5 tolO Vcs of 1% phosphoric acid- 1% Acetonitrile-98% Water to exchange the BF4 anions for phosphate anions. The column is then washed with 5 to 10 Vcs of 100 mM aqueous Guanidine.HCl to remove the phosphate anions and to exchange the phosphate anions to chloride anions. Finally the chloride anions are exchanged for acétate anions. The fractions containing the "puré product acétate salt" are combined, and the organic volátiles are removed under reduced pressure. The aqueous residue is lyophilized or precipitated after removal of water. The final API is analysed according to the USP/ EP Methods of Analysis.
Table 1
Comparison of the Surrogate Stationary Phase aided Prep-RP-HPLC with existing methods
13

Total Input (g) Output
Column ¡ (g)
volume (mL) | |
% Yield
4.0 g ¡1.2g
HPLC jColumn
Method I dimensions ¡(IDXL)
1 Standard RP- ¡ YMC, ODS-AQ
HPLC | [Comparativ
jeexample] ¡120 Á | diameter)
(50 mm X 250 ¡ mm, C18, 10 u, 490.0 pore;

Purity by ¡HPLC ¡
30.0% 199.86

Purification ' Waters Symmetry;
: method (19 mm X 50 mm,
14.2
[Example-1 C8, 5u, 120 Á depicting the i pore diameter) Present ¡ Invention] ¡
i Purification ¡ Discovery Bio
i method Wide Pore (10 mm j
| [Example-1 IX 250 mm, C18, 51 ,
i depicting the; u, 300Á pore i
Present ! diameter)
! Invention] ¡ |

l-4g
|l-2g

0.42g
0.32 g

30.0% 199.79
■4-
99.73
26.7%

This table reveáis that loadings of 7 to 10 times capacity of conventional prep-RP-HPLC are achievable with the processes described in the present invention.
14

Examples
Example-1: Preparative RP-HPLC of Leuprolide Acétate:
The C-18/ C-8 reversed phase column was pre-equilibrated with 5 to 10 column volumes (Vcs) of 5 to 10% aqueous acetonitrile containing 10 mM TBAHS. Two different columns were evaluated for the purification of Leuprolide: A Waters Symmetry Column {column parameters: 19 mm (Internal Diameter, ID) X 50 mm (Length, L), C8, 5u particles, 120 Á pore diameter, Amount loaded was 1.4 g of crude Leuprolide} and a Discovery Bio Wide Pore column {column parameters: 10 mm (ID) X 250 mm (L), C18, 5 u particles, 300Á pore diameter, Amount loaded was 1.2 g of crude Leuprolide ) were used. The column was pre-equilibrated with 5 to 10 column volumes (Vcs) of 10 mM TBAHS in 10% aqueous acetonitrile. After the loading was complete, the column was washed with 2 Vcs of Buffer A. Next, the gradient elution process was started. The buffer B was 300 mM TBAHS in 10% aqueous acetonitrile. A linear gradient of 0%B to 100% Buffer B over 60 min. was used for elution. A gradient hold was applied until all the API has eluted from the column. The fractions containing the puré API product were combined and treated with 1.5 to 2 equivalents of sodium tetrafluoroborate (NaBF4) and extracted 3 times with chloroform. The entire purification process was repeated 3 times. The solution contained "puré Leuprolide" fractions was combined and loaded on to a C-18 column from which all the TBAHS had been removed as described before.
The conversión of phosphate/ hydrogen sulphate anions to acétate anions was done as described earlier. Fractions containing puré Leuprolide Acétate API were lyophilized. The purification yield was about 30%>. (TBAHS herein denotes tetra-butyl-ammonium hydrogen sulphate)
Example-2: Preparative RP-HPLC of Triptorelin Acétate
The C-18/ C-8 reversed phase column was pre-equilibrated with 5 to 10 column volumes (Vcs) of 5 to 10%o aqueous acetonitrile containing 10 mM TBAHS. A Discovery Bio Wide Pore column {column parameters: 10 mm (ID) X 250 mm (L), C18, 5 u particles, 300Á pore diameter, Amount loaded was 1.0 g of crude Triptorelin} was used. The column was pre-equilibrated with 5 to 10 column volumes (Vcs) of 10 mM TBAHS in 10%) aqueous acetonitrile. After the loading was complete, the column was washed with 2 Vcs of Buffer A. Next, the gradient elution process was started. The buffer B was 300 mM TBAHS in 10% aqueous acetonitrile. A linear gradient of 0%B to 100%) Buffer B over 60 min. was used for elution. A gradient hold was applied until all the API has eluted from the column. The fractions containing the puré API product were
15

combined and treated with 1.5 to 2 equivalents of sodium tetrafluoroborate (NaBF^ and extracted 3 times with chloroform. The entire purification process was repeated 3 times. The solution contained "puré Triptorelin" fractions was combined and loaded on to a C-18 column from which all the TBAHS had been removed as described before.
The conversión of phosphate/ hydrogen sulphate anions to acétate anions was done as described earlier. Fractions containing puré Triptorelin API were lyophilized. The purification yield was about 25%. (TBAHS herein denotes tetra-butyl-ammonium hydrogen sulphate)
Example-3: Preparative RP-HPLC of Leuprolide Acétate employing n-Tetrabutylammonium bromide (TBA-Br):
The C-18 reverse phased column [Grace Vydac Column with column parameters 12 gms of C-18, 40 microns particles, 60Á pore diameter] was saturated with solution of 36 gms of TBA-Br in 360 mL of water at the flow rate of 8.0 ml/min. The column was then equilibrated with Buffer A (25mM TBA-Br in water) about 10 column volumes at the flow rate of 8.0 ml/min. The crude leuprolide Trifluoroacetate salt was dissolved in Buffer A and loaded on to the column. After the loading is complete, the gradient elution process was started. The Buffer B is 25mM of TBA-Br in 50% aqueous acetonitrile. A linear gradient of 0% of Buffer B to 100% of Buffer B over 10 Column volumes was applied. When Leuprolide is about to elute, a gradient hold may be applied until all the API has eluted from the column. The fraction containing the puré Leuprolide are combined after confírming the purity on an analytical HPLC. Percentage yield: 66.4%. Herein TBA-Br is n-Tetrabutylammonium bromide.
Removal of TBA-Br from the C-18 column: The column was first washed with at least 5 column volumes of 0.1M sodium tetrafluoroborate in acetonitrile and water (8:2).
Example-4: Preparative RP-HPLC of Leuprolide Acétate employing n-tetrabutylammoniutn hydrogen sulfate (TBA-HS)
The C-18 reverse phased column [Grace Vydac Column with column parameters 12 gms of C-
18, 40 microns particles, 60Á pore diameter] was saturated with solution of 36 gms of TBA-HS
in 360 mL of water at the flow rate of 8.0 ml/min. The column was then equilibrated with Buffer
A (25mM TBA-HS in water) about 10 column volumes at the flow rate of 8.0 ml/min. The crude
16

leuprolide Trifluoroacetate salí was dissolved in Buffer A and loaded on tó the column. After the loading is complete, the gradient elution process was started. The Buffer B is 25mM of TBA-HS in 50% aqueous acetonitrile. A linear gradient of 0% of Buffer B to 100% of Buffer B over 10 Column volumes was applied. When Leuprolide is about to elute, a gradient hold may be applied until all the API has eluted from the column. The fraction containing the puré Leuprolide are combined after confirming the purity on an analytical HPLC. Percentage yield: 64.4%. Herein TBA-HS is n-Tetrabutylammonium sulfate.
Remo val of TBA-HS from the C-18 column: The column was first washed with at least 5 column volumes of 0.1M sodium tetrafluoroborate in acetonitrile and water (8:2).
Example-5: Preparative RP-HPLC of Leuprolide Acétate employing Cetyltrimethylammonium bromide (CTA-Br)
The C-18 reverse phased column [Grace Vydac Column with column parameters 12 gms of C-18, 40 microns particles, 60Á pore diameter] was saturated with solution of lmM CTA-Br in water at the flow rate of 8.0 ml/min. The column was then equilibrated with Buffer A (5mM CTA-Br in water) about 10 column volumes at the flow rate of 8.0 ml/min. The crude leuprolide Trifluoroacetate salt was dissolved in Buffer A and loaded on to the column. After the loading is complete, the gradient elution process was started. The Buffer B is 5mM of CTA-Br in 50% aqueous acetonitrile. A linear gradient of 0% of Buffer B to 100% of Buffer B over 10 Column volumes was applied. When Leuprolide is about to elute, a gradient hold may be applied until all the API has eluted from the column. The fraction containing the puré Leuprolide are combined after confirming the purity on an analytical HPLC. Percentage yield: 61.4%. Herein CTA-Br is Cetyltrimethylammonium bromide.
Remo val of CTA-Br from the C-18 column: The column was first washed with at least 5 column volumes of 0.1M sodium tetrafluoroborate in acetonitrile and water (8:2).
Example-6: Preparative RP-HPLC of Leuprolide Acétate employing n-Tetrabutylphosphonium chloride (TBP-C1)
The C-18 reverse phased column [Grace Vydac Column with column parameters 12 gms of C-
18, 40 microns particles, 60Á pore diameter] was saturated with solution of 36 gm of TBP-C1 in
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360 mi of water at the flow rate of 8.0 ml/min. The column was then equilibrated with Buffer A (25mM TBP-C1 in water) about 10 column volumes at the flow rate of 8.0 ml/min. The crude leuprolide Trifluoroacetate salt was dissolved in Buffer A and loaded on to the column. After the loading is complete, the gradient elution process was started. The Buffer B is 25mM of TBP-C1 in 50% aqueous acetonitrile. A linear gradient of 0% of Buffer B to 100% of Buffer B over 10 Column volumes was applied. When Leuprolide is about to elute, a gradient hold may be applied until all the API has eluted from the column. The fraction containing the puré Leuprolide are combined after confirming the purity on an analytical HPLC. Percentage yield: 60.3%. Herein TBP-C1 is n-Tetrabutylphosphonium chloride.
Removal of TBP-C1 from the C-18 column: The column was first washed with at least 5 column volumes of 0.1M sodium tetrafluoroborate in acetonitrile and water (8:2).
Example-7: Preparative RP-HPLC of Leuprolide Acétate employing n-Tetrabutylammonium chloride (TBA-Cl)
The C-18 reverse phased column [Grace Vydac Column with column parameters 12 gms of C-18, 40 microns particles, 60Á pore diameter] was saturated with solution of 36 gm of TBA-Cl in 360 mi of water at the flow rate of 8.0 ml/min. The column was then equilibrated with Buffer A (25mM TBA-Cl in water) about 10 column volumes at the flow rate of 8.0 ml/min. The crude leuprolide Trifluoroacetate salt was dissolved in Buffer A and loaded on to the column. After the loading is complete, the gradient elution process was started. The Buffer B is 25mM of TBA-Cl in 50% aqueous acetonitrile. A linear gradient of 0% of Buffer B to 100%) of Buffer B over 10 Column volumes was applied. When Leuprolide is about to elute, a gradient hold may be applied until all the API has eluted from the column. The fraction containing the puré Leuprolide are combined after confirming the purity on an analytical HPLC. Percentage yield: 53.5%). Herein TBA-Cl is n-Tetrabutylammonium chloride.
Removal of TBA-Cl from the C-18 column: The column was first washed with at least 5 column volumes of 0.1M sodium tetrafluoroborate in acetonitrile and water (8:2).